CaM kinase IV is a Ca2+/calmodulin (CaM)-dependent protein kinase selectively expressed in T lymphocytes which was recently found to exhibit a dramatic increase in kinase activity within seconds following T cell stimulation through the T cell receptor. When overexpressed, a constitutively active form of the enzyme was shown to increase IL-2 expression 3-4-fold in response to stimulation of the cells. These data strongly implicate CaM kinase IV as a key component of Ca2+-mediated signaling during T cell activation. It is the long range goal of this project to understand how the Ca2+ signal during T lymphocyte activation. Preliminary results show that activation of recombinant rat brain CaM kinase IV requires Ca2+/CaM and phosphorylation of CaM kinase IV by a protein kinase activator. The activator will be cloned from a human T cell cDNA library. In addition, human T cell lymphocyte CaM kinase IV mutants will be used to identify the activating phosphorylation site/sites and to investigate the role Ca2+/CaM during activation. The second specific goal will be to determine how CaM kinase IV activates IL- 2 expression. The transcription factors CREB and Etsl, and the protein phosphatase, calcineurin, will be tested as downstream targets of CaM kinase IV. If necessary, and if time permits, a second approach to identify physiological targets will be taken. T cell proteins that directly interact with CaM kinase IV will be identified using the yeast two-hybrid system, affinity chromatography, and the gell shift assay and subsequently tested as substrates of the enzyme in vitro and in vivo.